characterizing the interaction between trace metal … the interaction between trace metal and...
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Characterizing the Interaction between Trace Metal and Dissolved Organic
Matter from the Florida Coastal Everglades
Rudolf Jaffé and Youhei Yamashita
Southeast Environmental Research Center & Department of Chemistry and Biochemistry,
Florida International University
Dissolved organic matter (DOM) affects the trace metal speciation
Mn+(H2O)n
ML
+ L
MX
+ X
(X = Cl, OH, CO3) (L = L1, L2….)
adsorption
M-colloid M-particle
adsorption
desorption
coagulation
disaggregation
Soil / Sediment
soluteexchange
resuspentionsettling
Organisms
biologicaluptake
regeneration
modified from Santschi et al., 1997
� Evaluation of functional groups of organic ligands✓Nuclear magnetic resonance (NMR)
✓Extended X-ray absorption fine structure (EXAFS)
etc
� Evaluation of complexing capacities of organic ligands
Chemical characterization of metal ion binding properties of DOM
� Evaluation of complexing capacities of organic ligands✓Electrochemical titration
✓Fluorescence quenching titration
✓Ion exchange
✓Competitive ligand exchange with solid-phase extraction
etc
However, these methods can determine “average”
metal ion binding properties of DOM.
Fluorescence quenching titration
120
130
140
150
Flu
ore
scence Inte
nsity
at E
x/E
m =
320/4
20
80
90
100
110
0 20 40 60 80
Total added metal conc. (10-6 M)
Flu
ore
scence Inte
nsity
at E
x/E
m =
320/4
20
360
380
400
420
440
0 50 100 150 200
Excita
tio
n (
nm
) Flu
ore
scence
Inte
nsity
(QS
U)
Excitation-Emission Matrix (EEM)
of surface water at SRS2
80
80
90
100
110
120
130
140
150
0 20 40 60
Total added metal conc. (10-6 M)
Flu
ore
scence Inte
nsity
at E
x/E
m =
320/4
20
300 350 400 450 500
260
280
300
320
340
360 200 250 300 350
Emission (nm)
Excita
tio
n (
nm
) Flu
ore
scence
Inte
nsity
(QS
U)
340
360
380
400
420
440
-175-150-125-100-75-50-25
Excita
tio
n (
nm
)
Ch
an
ge
s in
flu
ore
scence
Changes in EEM of SRS2 water
with 70µM Cu(II)
300 350 400 450 500
26
0
280
300
320
340 -25 0
Emission (nm)
Excita
tio
n (
nm
)
Ch
an
ge
s in
flu
ore
scence
EEM is useful for determining the binding capacities of each
type of the fluorescence components with trace metals.
However, peak picking technique is problematic for quantitative estimation.
PARAFAC statistically decompose EEMs into independent fluorescent group
Parallel factor analysis (PARAFAC)
Observed EEM Modeled EEM
� Reproducibility
To test the availability and sensitivity of combination technique of fluorescence quenching titration and EEM-PARAFAC
Purposes of the present study
� Differences in binding capacities among different fluorescent components / DOM in different sites / different trace metals
Materials and Methods
✓Surface water samples 4 FCE-LTER sites
●SRS2
●SRS4
●SRS6
●TS2
SRS2
SRS4SRS6 TS2
✓Trace metals ●Cu(II): semi-hard metal
●Hg(II): soft metal
110
120
130
140
150
Flu
ore
scence Inte
nsity (
QS
U)
MMLM
LM
LM CKCKCK
IIII ++−+= 1)(2
1)(( 00
Ryan and Weber Model
)4)1(22
MLMMMLM CCKCKCK −++−
I: fluorescence intensity at the metal conc. C
Materials and Methods
Modeling of fluorescence quenching curves
80
90
100
110
0 20 40 60 80
Total added metal conc. (10-6 M)
Flu
ore
scence Inte
nsity (
QS
U)
100)(
0
0×
−=
I
IIf ML
I: fluorescence intensity at the metal conc. CM
I0: fluorescence intensity without metal
IML: fluorescence intensity which dose not
change due to the addition of metal
KM: conditional stability constant
CL: total ligand concentration
f: fraction of the initial fluorescence that corresponds to the binding fluorophores
Materials and Methods
PARAFACE modelingE
xcita
tio
n (
nm
)
300
400
300 400 500
Component 1Terrestrial humic
400
Component 4Microbial humic
300
400
300 400 500
Component 3Terrestrial humic
300
400
300 400 500
Component 2Terrestrial humic
400
Component 5Microbial humic
400
Component 6Terrestrial humic
Excita
tio
n (
nm
)
300
300 400 500
300
300 400 500
300
300 400 500
300
400
300 400 500
Component 7Protein
300
400
300 400 500
Component 8Protein
Excita
tio
n (
nm
)E
xcita
tio
n (
nm
)
Emission (nm) Emission (nm)
Emission (nm)
� 1108 surface water samples from Florida Coastal Everglades were used for FCE-PARAFAC model.
� Source characterization was carried out by comparison to previous PARAFAC studies (Cory and Mcknight, 2005; Stedmon and Markager, 2005; Yamashita et al., 2008).
160
180
90
95
22
24
Reproducibility
Triplicate titration experiments with Cu(II) for SRS2 water
Flu
ore
sce
nce in
ten
sity (
QS
U)
300
400
300 400 500
Component 1Terrestrial humic
300
400
300 400 500
Component 4Microbial humic
300
400
300 400 500
Component 7Protein
logK = 4.83±0.03f = 46±1
logK = 5.06±0.04f = 29±1
80
100
120
140
160
0 10 20 30 40 50 60 7060
65
70
75
80
85
0 10 20 30 40 50 60 7010
12
14
16
18
20
0 10 20 30 40 50 60 70
Flu
ore
sce
nce in
ten
sity (
QS
U)
Cu(II) (µM) Cu(II) (µM) Cu(II) (µM)
1st titration2nd titration3rd titration
The combination of fluorescence quenching titration and EEM-PARAFAC is enough to reproducibly determine the binding capacity of individual humic-like components with trace metals.
Differences in quenching among DOM/trace metals
Titration experiments with Cu(II) or Hg(II) for 4 different samples
90
100
90
100
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
300
400
300 400 500
Component 2Terrestrial humic
40
50
60
70
80
0 10 20 30 40 50 60 7040
50
60
70
80
0 10 20 30 40 50 60 70
Cu(II) (µM) Hg(II) (µM)
F/F
0×
10
0
TaylorSloughSRS2
SRS4SRS6TS2
Differences in quenching among fluorescent components
Titration experiments with Cu(II) for SRS2 water
Comp1 (Terrestrial humic)Comp2 (Terrestrial humic)Comp3 (Terrestrial humic)Comp4 (Microbial humic)
F/F
0×
10
070
80
90
100
Comp4 (Microbial humic)Comp5 (Microbial humic)Comp6 (Terrestrial humic)
Cu(II) (µM)
F/F
0
40
50
60
0 10 20 30 40 50 60 70
SRS2
Fluorescence quenching titration with EEM-PARAFAC is a sensitive method to determine the differences in binding capacity of individual fluorescent components with trace metals.
The logK and f values of 6 humic-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS4
SRS6
TS2TS2
The logK and f values of 6 humic-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS4
SRS6
TS2
0
0
6.524
TS2
The logK and f values of 6 humic-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS4
SRS6
TS2
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg70
60
50
70
60
50
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg
0
0
6.524
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
The logK and f values of 6 humic-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS4
SRS6
TS2
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg70
60
50
70
60
50
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg
0
0
6.524
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
The logK and f values of 6 humic-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS4
SRS6
TS2
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg70
60
50
70
60
50
70
60
50
70
60
50
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
SRS2SRS4SRS6TS2
(A) Cu (B) Hg
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
f(%
) 40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
40
30
20
10
0Comp1 Comp2 Comp3 Comp4 Comp5 Comp6
no
t m
od
ele
d
no
t m
od
ele
d
Changes in fluorescence of protein-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS490
100
110
120
90
100
110
120
300
400
300 400 500
Component 7Protein
Titration experiments with Cu(II) or Hg(II) for 4 different samples
Cu(II) (µM) Hg(II) (µM)
SRS6TS2
50
60
70
80
90
0 10 20 30 40 50 60 7050
60
70
80
90
0 10 20 30 40 50 60 70
Changes in fluorescence of protein-like components
SRS6
SRS4
SRS2
TS2
Shark RiverSlough
TaylorSloughSRS2
SRS490
100
110
120
90
100
110
120
300
400
300 400 500
Component 7Protein
Titration experiments with Cu(II) or Hg(II) for 4 different samples
Cu(II) (µM) Hg(II) (µM)
SRS6TS2
50
60
70
80
90
0 10 20 30 40 50 60 7050
60
70
80
90
0 10 20 30 40 50 60 70
Increases in fluorescence intensity at the later stage of the Cu(II) addition experiment might be the result of:
� Changes in quantum yields by changes in 3D-structure of protein-molecules due to high concentrations of Cu(II).
� Changes in quantum yields of protein molecules from a shift of protein-inorganic complexes to protein-Cu(II) complexes.
� Release of protein molecules due to replacement from DOM-protein interaction to DOM-Cu(II) interactions.
Summary
� The combination of fluorescence quenching titration and EEM-PARAFAC provides adequate reproducibility and sensitivity for the determination of the binding capacity of individual humic-like components with trace metals.
� The trace metal binding capacity and behavior was � The trace metal binding capacity and behavior was different among humic-like components determined by PARAFAC.
� The changes in protein-like fluorescence intensity with Cu(II) additions indicate that EEM-PARAFAC is also effective in evaluating the changes in molecular environments of DOM, like as DOM-DOM interactions.
Acknowledgements
� FCE-LTER
� College of Arts and Science at FIU for financial support
� The Wetlands Ecosystem Laboratory at SERC for logistic support
� Geochemistry Group at SERC for support to Lab work